Hydroxytyrosol-rich composition from olive vegetation water and method of use thereof

ABSTRACT

The invention provides olive-derived hydroxytyrosol. According to one aspect of the invention, vegetation water is collected from olives. Acid is added to stabilize the vegetation water and prevent fermentation. The mixture is incubated to allow oleuropein to convert to hydroxytyrosol, and then fractionated to separate hydroxytyrosol from other components. The hydroxytyrosol is useful as a therapeutic and anti-oxidant for a variety of health purposes, including for the treatment of skin damage. In addition, the hydroxytyrosol is useful as a natural anti-bacterial, anti-viral and fungicidal product for agricultural and pest control applications.

This application is a continuation of U.S. patent application Ser. No.10/190,043, filed on Jul. 5, 2002 and issued May 11, 2010 as U.S. Pat.No. 7,713,569. application Ser. No. 10/190,043 claims priority benefitto U.S. provisional application 60/356,847, filed Feb. 13, 2002, and isa continuation-in-part of U.S. patent application Ser. No. 09/944,744,filed on Aug. 31, 2001, now issued as U.S. Pat. No. 6,416,808, and thecorresponding PCT application number PCT/US01/27132, both filed Aug. 31,2001, both of which claim priority to U.S. provisional application60/230,535, filed Sep. 1, 2000. Each of the aforementioned isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to a phenolic fraction of a group of compoundspresent in the fruit and leaves of olive plants, which are known asPolyphenols. Particularly, the invention provides an olive extractcontaining hydroxytyrosol (3,4-dihydroxyphenylethanol), with low amountsor substantially free of oleuropein and tyrosol, and a method ofobtaining the same and to methods of use of such compounds.

REFERENCES

Armstrong, B. K. and Doll, R., International. J. Cancer 15:617-631(1975).

Bartsch, H., et al., Carcinogenesis 20:2209-2218 (1999).

Braga, C., et al., Cancer 82:448-453 (1998).

Chan, J. M., et al., Seminars in Cancer Biology 8:263-273 (1998).

d'Amicis, A. and Farchi, S., in: Advances in Nutrition and Cancer 2(Zappia, V., et al., Eds.) 67-72, Kluwer Academic/Plenum Publishers, NewYork (1999).

Deiana, M., et al., Free Radic. Biol. Med. 26:762-769 (1999).

de la Puerta, R., et al., Biochem. Pharmacol. 57:445-449 (1999).

Ficarra, P., et al., Farmaco 46:803-815 (1991).

Gerber, M., Epidemiology of Diet and Cancer, ed. M. J. Hill, 263-275(1994).

Kohyama, N., et al., Biosci. Biotechnol. Biochem. 61:347-350 (1997).

Kuller, L. H., Journal of the American Dietetic Association 97:S9-S15(1997).

La Vecchia, C., et al., European Journal of Cancer Prevention 7:461-464(1998).

Manna, C., et al., FEBS Letters 470:341-344 (2000).

Martin-Moreno, J. M., et al., Int. J. Cancer 58:774-780 (1994).

Mattson, F. H. and Grundy, S. M., J. Lipid Res. 26:194-202 (1985).

Owen, R. W., et al., J. Can. Res. Clin. Onc. 125:S31 (2000a).

Owen, R. W., et al., Eur. J. Cancer 36:1235-1247 (2000b).

Owen, R. W., et al., Food Chem. Toxic. 38:647-659 (2000c).

Parthasarathy, S., et al., Proc. Natl. Acad. Sci. USA 87:3894-3898(1990).

Petroni, A., et al., Thromb. Res. 78:151-160 (1995).

Risch, H. A., et al., Journal of the National Cancer Institute86:1409-1415 (1994).

Romani, A., et al., J. Agric. Food Chem. 47:964-967 (1999).

Tsimidou, M., et al., Food Chem. 44:53-60 (1992).

Visioli, F., et al., FEBS Letters 468:159-160 (2000).

Visioli, F. and Galli, C., Nutr. Rev. 56:142-147 (1998).

BACKGROUND OF THE INVENTION

A high amount of dietary fat has been implicated in the development ofseveral diseases (Owen et al., 2000c). Atherosclerosis (Kuller, 1997)and coronary heart disease (Gerber, 1994), as well as cancer of thebreast (La Vecchia et al., 1998), prostate (Chan et al., 1998), ovary(Risch et al., 1994), and colon (Armstrong and Doll, 1975) have eachbeen associated with elevated dietary fat. However, evidence indicatesthat it is not only the amount, but also the type of dietary fat that isimportant in the etiology of some cancers (Bartsch et al., 1999).

Olive oil, the principal fat component of the Mediterranean diet, hasbeen associated with a lower incidence of coronary heart disease (Owenet al., 2000b; Parthasarathy et al., 1990; Mattson and Grundy, 1985) andcertain cancers (d′Amicis and Farchi, 1999; Braga et al., 1998;Martin-Moreno et al., 1994). Several laboratories have reported that thehydrolysis of the olive oil phenolics oleuropin and other family memberslead to small phenolic components with strong chemoprotective activity(Owen et al., 2000a; Manna et al., 2000). In particular, the olive oilphenolic hydroxytyrosol prevents low density lipoprotein (LDL) oxidation(Visioli and Galli, 1998), platelet aggregation (Petroni et al., 1995),and inhibits 5- and 12-lipoxygenases (de la Puerta et al., 1999; Kohyamaet al., 1997). Hydroxytyrosol has also been found to exert an inhibitoryeffect on peroxynitrite dependent DNA base modification and tyrosinenitration (Deiana et al., 1999), and it counteracts cytotoxicity inducedby reactive oxygen species in various human cellular systems (Manna etal., 2000). Finally, studies have shown that hydroxytyrosol isdose-dependently absorbed in humans following ingestion, indicating itsbioavailability (Visioli et al., 2000).

Conventionally, olive oil production involves crushing olives, includingthe pits, to produce a thick paste. During this procedure, the crushedolives are continuously washed with water, a process known as“malaxation.” The paste is then mechanically pressed to squeeze out theoil content. In addition to providing olive oil, the pressing alsosqueezes out the paste's water content. Such washing and pressing stepsyield a considerable amount of water, referred to as “vegetation water.”

Both the pit and the pulp of olives are rich in water-soluble, phenoliccompounds. Such compounds are extracted from olives during malaxation,according to their partition coefficients, and end up in the vegetationwater. This explains why various phenolic compounds, such as oleuropeinand its derivatives, produced in olive pulp, can be found in abundancein vegetation waters. Similarly, a number of monophenolic compounds,such as tyrosol and its derivatives, produced in olive pits, are alsoabundant in vegetation waters.

Because of the strong chemoprotective activity of hydroxytyrosol, it isdesirable to develop a method which produces an aqueous olive extractwith a high percentage of hydroxytyrosol.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a method of producing ahydroxytyrosol-rich composition. The method has the steps of (a)producing vegetation water from olives, preferably from the meat (orpulp) of depitted olives, (b) adding acid to the vegetation water,preferably, in an amount to produce a pH between 1 and 5, and morepreferably between 2 and 4, and (c) incubating the acidified vegetationwater until at least 50%, preferably at least 75%, and more preferablyat least 90% of the oleuropein originally present in the vegetationwater has been converted to hydroxytyrosol. In a preferred embodiment,the acidified vegetation water is incubated for a period of at least twomonths, and even more preferably, the acidified vegetation water isincubated up to a period of approximately between 6-12 months.

In one embodiment, the incubating is carried out until the vegetationwater has a weight ratio of hydroxytyrosol to oleuropein of between 1:1and 200:1, preferably 4:1 and 200:1, and more preferably 10:1 and 100:1.In a related embodiment, the incubating is carried out until thevegetation water has a weight ratio of hydroxytyrosol and tyrosol ofbetween 3:1 and 50:1, typically 5:1 to 30:1.

The method may further include fractionating the incubated, vegetationwater to separate hydroxytyrosol from other components, and/or dryingthe vegetation water rich in hydroxytyrosol to produce a dried extract.In one embodiment, the incubated vegetation water is extracted with anorganic solvent to produce a 20%, or preferably 95% or more richfraction in hydroxytyrosol.

Also provided is an injectable composition that includes ahydroxytyrosol-rich composition prepared by one or more of theembodiments described above.

In another aspect, the invention includes a method of producing ahydroxytyrosol-rich composition that includes the steps of (a) producingvegetation water from olives; (b) hydrolyzing the oleuropein and otherlarge phenolic molecules by addition of acid (c) optionally, drying thevegetation water; (d) contacting the optionally dried vegetation waterwith a supercritical fluid; and (e) recovering the hydroxytyrosol-richcomposition from the contacted vegetation water. In one embodiment, thehydroxytyrosol-rich composition includes at least about 95 percent byweight hydroxytyrosol. In another embodiment, the hydroxytyrosol-richcomposition includes at least about 97 percent by weight hydroxytyrosol.In yet another embodiment, the hydroxytyrosol-rich composition includesat least about 99 percent by weight hydroxytyrosol.

In another aspect, a method of producing a hydroxytyrosol-richcomposition that includes the steps of (a) producing vegetation waterfrom olives; (b) hydrolyzing the oleuropein and other large phenolicmolecules by addition of acid (c) optionally, drying the vegetationwater; (d) extracting the vegetation water with a suitable organicsolvent, such as Ethyl Acetate (EtAc); and (e) recovering a fractionthat contains hydroxytyrosol in a purity equal or higher than 95% of thetotal phenolic fraction. In one embodiment, the hydroxytyrosol-richcomposition includes at least 20% of a phenolic fraction containingabout 95 percent by weight hydroxytyrosol. In one embodiment, the EtAcfraction is purified by silica gel chromatography or other gelchromatography to obtain an hydroxytyrosol fraction containing 95% ormore by weight hydroxytyrosol.

In one embodiment, the recovering step described above includes thesteps of (a) recovering the supercritical fluid, where the supercriticalfluid contains the hydroxytyrosol; and (b) vaporizing the supercriticalfluid to extract the hydroxytyrosol-rich composition. In anotherembodiment, the contacting step described above comprises the steps of(a) providing a porous membrane having opposite sides in a module underpressure with the membrane serving as a barrier interface between afluid and a dense gas, the membrane being nonselective for saidhydroxytyrosol; (b) providing the supercritical fluid into the module onone side of the membrane and the vegetation water on the opposite sideof the membrane; (c) and extracting the hydroxytyrosol across themembrane as driven by a concentration gradient of the hydroxytyrosolbetween the vegetation water and the supercritical fluid. In oneembodiment, the porous membrane is a hollow fiber membrane. In anotherembodiment, the supercritical fluid is carbon dioxide.

In another embodiment, the present invention comprises a method ofproducing a hydroxytyrosol-rich composition that includes the steps of(a) producing vegetation water from olives; (b) hydrolyzing theoleuropein and other large phenolic molecules by addition of acid; and(c) spray drying, i.e., evaporating the acidified vegetation waterthereby resulting in a powder containing hydroxytyrosol. In anotherembodiment, the evaporation step described above is performed by theaddition of maltodextrins to the acidified vegetation water topreferably result in a powder containing approximately 1 to 5%hydroxytyrosol, and more preferably, a powder containing approximately2% hydroxytyrosol.

In another aspect, the invention includes a dietary supplementcomprising an aqueous extract of olives containing a weight ratio ofhydroxytyrosol to oleuropein of between 4:1 and 200:1, typically 10:1and 100:1.

In a related aspect the invention includes a dietary supplementcomprising an aqueous extract of olives containing a weight ratio ofhydroxytyrosol and tyrosol of between 3:1 and 50:1, typically 5:1 and30:1.

The above supplements may be dried, preferably by spray drying, toprovide a powder extract, which can formulated into a tablet, capsule,pill, or confection food additive. Alternatively, the above supplementsmay be incorporated in a pharmaceutical formulations such as into ahydroxytyrol-rich injectable formulation.

Also provided are methods of protecting skin against adverse effects ofexposure to ultaviolet radiation (UVR) comprising administering to asubject in need of such protection a pharmaceutically effective amountof a treatment agent having a weight ratio of hydroxytyrosol tooleuropein of between about 1:1 and about 200:1, preferably betweenabout 4:1 and about 100:1, and more preferably between about 10:1 andabout 50:1. The agent may also include a sunscreen for topicalapplications. In one embodiment, the agent is administered topically.Preferably, the agent is administered orally.

These and other aspects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying figures and tables.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the structures of phenolic compounds and their precursorsdetected in olive oil: ligstroside (I); oleuropein glucoside (II);aglycone of ligstroside (III); aglycone of oleuropein glucoside (IV);dialdehydic form of ligstroside aglycone laking a carboxymethyl group(V); dialdehydic form of oleuropein glucoside aglycone lacking acarboxymethyl group (VI); tyrosol (VII); hydroxytyrosol (VIII).

FIG. 2 shows the HPLC analysis of a hydroxytyrosol-rich composition ofthe invention after supercritical carbon dioxide extraction fromvegetation water obtained from the meat of depitted olives.

FIG. 3 shows the HPLC analysis of a hydroxytyrosol-rich composition ofthe invention following supercritical carbon dioxide extraction, withsynthetic hydroxytyrosol.

FIG. 4 shows the HPLC analysis of a hydroxytyrosol-rich composition ofthe invention after acidic hydrolysis of vegetation water obtained fromthe meat of depitted olives.

FIG. 5 shows the HPLC analysis of a hydroxytyrosol-rich composition ofthe invention following ethyl acetate extraction of hydroxytyrosol fromvegetation water obtained from depitted olives and hydrolyzed by acidaddition.

FIG. 6 shows the HPLC analysis of pure (95% or more) hydroxytyrosolobtained after purification by gel chromatography on silica gel.

FIG. 7 shows the mass spectrum of a hydroxytyrosol-rich composition ofthe invention.

FIG. 8 illustrates the fragmentation pathway of hydroxytyrosol.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents, patent applications or other references citedin this application are herein incorporated by reference in theirentirety as if each individual publication, patent, patent applicationor reference are specifically and individually indicated to beincorporated by reference.

Definitions

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art of the present invention. It isto be understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary.

By “oleuropein” is intended secoiridoid glucoside oleuropein (StructureII in FIG. 1).

By “tyrosol” is intended 4-hydroxyphenethyl alcohol (Structure VII inFIG. 1).

By “hydroxytyrosol” is intended 3, 4-dihydroxyphenethyl alcohol(Structure VIII in the FIG. 1).

Method of the Invention

The invention provides, in one aspect, provides a hydroxytyrosol-richcomposition from olive-derived vegetation water. It has been discoveredthat under specific conditions, as described below, hydroxytyrosol maybe obtained from the vegetation water of olives. Considered below arethe steps in practicing the invention.

Producing Vegetation Water

The method of the invention employs olives that may be obtained fromconventional and commercially available sources such as growers.Preferably, the vegetation water is obtained from pitted olives. Theolives processed according to the method disclosed herein may be pittedby any suitable means. Pits in the olives contain tyrosol which is anundesired component in the vegetation water and which may not beappreciably broken down by the acid treatment described below. The pitsmay be separated from the pulp manually or in an automated manner asdescribed below. Preferably, such means should be capable of segregatingthe pits without breaking them, which might otherwise cause higherconcentrations of tyrosol in the vegetation water. In anotherembodiment, hydroxytyrosol is extracted from vegetation water obtainedfrom olives that have not been pitted.

To produce vegetation water, olive pulp from the olives is first pressedto obtain a liquid-phase mixture including olive oil, vegetation water,and solid by-products. Thereafter, the vegetation water is separatedfrom the rest of the liquid phase mixture and collected. Exemplarymethods of obtaining vegetation water are described in co-owned U.S.Patent Application Nos. 6,165,475 and 6,197,308, both to R. Crea, eachof which are expressly incorporated herein by reference in theirentirety.

For purposes of commercial production, it may be desirable to automatevarious aspects of the invention. In this regard, one embodimentcontemplates the use of an apparatus as disclosed in U.S. Pat. Nos.4,452,744, 4,522,119 and 4,370,274, each to Finch et al., and eachexpressly incorporated herein by reference. Briefly, Finch et al. teachan apparatus for recovering olive oil from olives. Initially, olives arefed to a pulper that separates the olive pits from the olives to obtaina pitless olive meat. The meat is then taken up by an extraction screwthat subjects the meat to an extraction pressure sufficient to withdrawa liquid phase, comprising oil, water and a minor proportion of olivepulp. The liquid phase is collected in a bin and then sent to aclarifying centrifuge that separates the pulp from the liquid phase toobtain a mixture comprising olive oil and vegetation water. A purifyingcentrifuge then separates the vegetation water and a small proportion ofsolid matter from the mixture to obtain an olive oil, substantially freeof vegetation water, that is collected in a tank. According to Finch etal., the water is put to a disposal means such as a sewer. The presentinvention, in sharp contrast, provides for the collection, saving anduse of the vegetation water to extract hydroxytyrosol.

Additional devices that may be used in practicing the present inventionare disclosed in Italian Patent Nos. 1276576 and 1278025, each of whichis expressly incorporated herein by reference. As above, these devicescan be used to separate the pulp from the pits prior to processing ofthe crushed olive pulp into oil, water, and solid residues.

Conversion of Oleuropein to Hydroxytyrosol

In one aspect of the invention, the oleuropein contained in thevegetation water is converted to hydroxytyrosol. The pH of thevegetation water may be decreased by the addition of acid, and thevegetation water allowed to incubate under conditions which, accordingto the discovery of the invention, promote acid hydrolysis of oleuropeinto hydroxytyrosol. The sample may then be fractionated or extracted toseparate hydroxytyrosol from other compounds.

In a preferred embodiment, the added acid is citric acid. The acid isadded to the vegetation water, preferably to adjust the pH to 1-5, andmore preferably, to a pH of 2-4. Solid citric acid can be added whilecontinuously stirring in an amount of preferably about 25 to 50 poundsof acid per about 1000 gallons of vegetation water. The pH of theresulting solution can be monitored, and further addition of acid may benecessary to achieve the desired pH. Exemplary methods showing theconversion of oleuropein to hydroxytyrosol following the addition ofcitric acid are given in Examples 1 and 2.

The acid may also be an organic or inorganic acid other than citricacid. Exemplary acids which may be used in the present invention includethe inorganic substances known as the mineral acids—sulfuric, nitric,hydrochloric, and phosphoric acids—and the organic compounds belongingto the carboxylic acid, sulfonic acid, and phenol (benzyl) groups. Theaddition of acid to the vegetation water serves several purposes: (i) itstabilizes the vegetation water from air (oxygen) polymerization ofphenolic molecules; (ii) it prevents fermentation of the vegetationwater by endogenous and/or exogenous bacteria and yeast; and (iii) itprovides for the hydrolysis of oleuropein and other large phenolicmolecules containing hydroxytyrosol, converting them intohydroxytyrosol, as shown in Examples 1 and 2. Tables 1 and 2, inExamples 1 and 2, respectively, contain data from two samples ofvegetation water and the respective percent composition of variouscomponents in the samples over time following the addition of citricacid. In one embodiment, the mixture is allowed to incubate untilhydroxytyrosol is 75-90% of the total combination of oleuropein andhydroxytyrosol. In another embodiment, substantially none of theoleuropein in the original mixture remains.

Purification of Hydroxytyrosol

Following the conversion of oleuropein to hydroxytyrosol, preferably byacid addition, the incubated vegetation water may be fractionated by anumber of methods known in the art. Exemplary methods of fractionationinclude partitioning with an organic solvent, such as Ethyl Acetate,chromatographic methods, including gel chromatography and high pressureliquid chromatography (HPLC), or supercritical fluids.

Alternatively, vegetation water obtained as described above afteracidification, provides a solution which is rich in low molecular weightpolyphenols, particularly hydroxytyrosol and a small amount of tyrosoland oleuropein. The concentration of hydroxytyrosol in the processedwater may range from 4-5 grams per liter to 10-15 grams per literdepending upon the degree of dilution by addition of water during theolive oil extraction. In one embodiment, the invention provides a methodof extraction or purification that selectively enriches the content ofhydroxytyrosol without the addition of contaminants. Thus, the majorpolyphenolic component, hydroxytyrosol, is isolated from other membersof the polyphenolic family, impurities, suspended solids, tannins, andother molecules contained in the vegetation water. Hydroxytyrosol maytherefore be produced in a purity and quantity not readily available bycurrent synthetic or natural extraction methods.

A supercritical fluid is a gas that becomes very dense above itscritical temperature and pressure. Its properties are between those of agas and liquid, resulting in increased ability to dissolve compounds.Its relatively high density, high diffusivity, and low viscosity allowit to extract compounds faster than conventional liquid solvents. Carbondioxide is the gas used most widely for supercritical fluid processingof foods and food ingredients because it is natural, nontoxic,non-flammable, and relatively inert and leaves no residue in theextracted product. Typical liquid extraction with supercritical carbondioxide is usually done by dispersing one phase in the other in largecontacting columns or towers, where the solute containing fluid, such asjuices, flows downward by gravity, and the supercritical carbon dioxideflows upward. Mass transfer occurs at the interface between the twophases.

Alternatively, continuous extraction of liquids and suspensions can beachieved using supercritical fluids, such as carbon dioxide, and porousmembranes instead of contacting columns. Instead of dispersing thephases, the liquid is fed continuously through porous polypropylenemembranes configured as hollow fiber bundles or spiral wound sheets. Theliquid passes through the porous membranes within a pressurized module,while supercritical carbon dioxide flows countercurrently on the otherside of the membrane. The pressure in the module is essentially thesame, so that the extraction is driven by the concentration gradientbetween the fluid and the supercritical carbon dioxide. The extract maybe recovered by vaporizing the carbon dioxide for recycling. Anexemplary method of extraction using supercritical carbon dioxide andporous membranes is described in U.S. Pat. No. 5,490,884, which isexpressly incorporated by reference herein in its entirety.

Other supercritical fluids, instead of, or in combination with, carbondioxide. These fluids include methane, ethane, propane, butane,isobutane, ethene, propene, hydrofluorocarbons, tetrafluoromethane,chlorodifluoromethane, carbon dioxide, dinitrogen monoxide, sulphurhexafluoride, ammonia, and methyl chloride.

Example 3 describes a small scale experiment in support of theinvention, wherein hydroxytyrosol was isolated from vegetation waterusing supercritical carbon dioxide and porous membranes. HPLC and massspectrometry analysis of the isolated hydroxytyrosol shows the sample tobe 97-99% pure hydroxytyrosol. Thus, the invention provides ahydroxytyrosol-rich composition containing at least about 80%hydroxytyrosol, preferably at least about 90% hydroxytyrosol, morepreferably at least about 95% hydroxytyrosol, even more preferably atleast about 97% hydroxytyrosol, and yet, even more preferably at leastabout 99% hydroxytyrosol.

Prior to extraction with a supercritical fluid the vegetation water mayhave carriers, which are known to those of skill in the art, such asmaltodextran and/or polypropylene beads, added to the solution; and/orthe solution may be dried. The drying step preferably removes at leastabout 90%, more preferably at least about 95%, and even more preferablyat least about 98% of the water from the vegetation water.

An important feature of membrane reactors is the fact that contactsurface interfacial area can be added independently of fluid velocities.Accordingly, the invention contemplates a large scale unit where thesurface membrane area of the membrane used for extraction is at leastabout 100 square yards, preferably at least about 300 square yards, andeven more preferably at least about 600 square yards to allow separationof hydroxytyrosol from large volumes of vegetation water. Thus, themembrane system of the invention would, in one aspect, be able toaccommodate a flow rate of between 1-20 liters per minute, preferablybetween 5-10 liters per minute.

Additional purification methods may also be used in accordance with theinvention as mentioned above. HPLC isolation of hydroxytyrosol isdescribed in: Ficarra et al., 1991; Romani et al., 1999; and Tsimidou,1992, each of which is expressly incorporated by reference herein.

Hydroxytyrosol-Rich Dietary Supplement

It should be appreciated that hydroxytyrosol produced by the methoddescribed above may be used for a variety of applications. For example,hydroxytyrosol obtained by the method of the present invention can beused: (i) as a natural anti-bacterial, anti-viral and/or fungicidalproduct for agricultural and/or pest control applications, and (ii) as atherapeutic and/or an anti-oxidant for a variety of health purposes. Inone exemplary embodiment, the hydroxytyrosol, is administered to amammalian subject, such as a person desirous of one or more of thebenefits associated with hydroxytyrosol.

Accordingly, provided herein are compositions and methods for theprotection of skin damage resulting from exposure to ultravioletradiation (UVR). The hydroxytyrosol obtained by the method of theinvention can be administered orally or parenterally. Oral dosage formscan be in a solid or liquid form. Such dosage forms can be formulatedfrom purified hydroxytyrosol or they can be formulated from aqueous oraqueous-alcoholic extracts. Regarding the latter, aqueous oraqueous-alcoholic (e.g., water or water-ethanol) extracts can bespray-dried to provide a dry powder that can be formulated into oraldosage forms with other pharmaceutically acceptable carriers. Theaqueous or aqueous-alcoholic extracts can be formulated to containvarious weight ratios of hydroxytyrosol to oleuropein of between 4:1 and200:1, preferably between about 10:1 and about 100:1. The extracts mayalso be formulated to contain various weight ratios of hydroxytysol andtyrosol of between about 2:1 and about 50:1, preferably between about5:1 and about 30:1.

Preferably, the composition is orally administered to a patient in needof protection against skin damage resulting from exposure to UVR. Thesolid oral dosage form compositions in accordance with this inventionare prepared in a manner well known in the pharmaceutical arts, andcomprise hydroxytyrosol in combination with at least onepharmaceutically acceptable carrier. In making such compositions, ahydroxytyrosol-rich composition, either in substantially pure form or asa component of a raw distillate or extract, is usually mixed, diluted orenclosed with a carrier. The carrier can be in a solid form, semi-solidor liquid material which acts as a vehicle, carrier or medium for theactive ingredient. Alternatively, the carrier can be in the form of acapsule or other container to facilitate oral administration. Thus, thesolid oral dosage forms for administration in accordance with thepresent invention can be in the form of tablets, pills, powders or softor hard gelatin capsules.

Alternatively, the hydroxytyrosol obtained in accordance with thisinvention for oral administration can be in liquid form wherein thepharmaceutically acceptable carrier is water or an aqueous-alcoholicmedium.

The compositions for administration in the present invention can also beformulated with other common pharmaceutically acceptable excipients,including lactose, dextrose, sucrose, sorbitol, mannitol, starches,gums, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, methylcellulose, water, alcohol and the like. Theformulations can additionally include lubricating agents such as talc,magnesium stearate and mineral oil, wetting agents, emulsifying andsuspending agents, preserving agents such as methyl- andpropylhydroxybenzoates, sweetening agents or flavoring agents. Further,the compositions of the present invention can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to a subject.

Parenteral formulations for use in accordance with the present inventionare prepared using standard techniques in the art. The term parenteralas used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection, or infusion techniques. Suchformulations are commonly prepared as sterile injectable solutions,using a parenterally acceptable carrier such as isotonic saline solutionor as a sterile packaged powder prepared for reconstitution with sterilebuffer or isotonic saline prior to administration to a subject. In onepreferred embodiment the parenteral formulation is an injectibleformulation which comprises between 1 and 500 mg/ml of thehydroxytyrosol rich composition of the present invention. Morepreferably, the injectible formulation comprises between 1 to 100 mg/mlof the hydroxytyrosol rich composition, even more preferably, between 10to 100 mg/ml of the hydroxytyrosol rich composition, and most preferablyabout 10 mg/ml of the hydroxytyrosol rich composition.

From the foregoing, it can be seen how various objects and features ofthe invention are met. Those skilled in the art can now appreciate fromthe foregoing description that the broad teachings of the presentinvention can be implemented in a variety of forms. Therefore, whilethis invention has been described in connection with particularembodiments and examples thereof, the true scope of the invention shouldnot be so limited. Various changes and modification may be made withoutdeparting from the scope of the invention, as defined by the appendedclaims.

The following examples illustrate methods of producinghydroxytyrosol-rich compositions in accordance with the invention. Theexamples are intended to illustrate, but in no way limit, the scope ofthe invention.

EXAMPLES Example 1 Conversion from Oleuropein to HydroxytyrosolFollowing the Addition of About 25 Pounds of Citric Acid/1000 Gallons

Table 1 shows the conversion of oleuropein to hydroxytyrosol over timefollowing the addition of about 25 pounds of citric acid per 1000gallons of vegetation water. The percentages in Table 1 are shown asweight percentages of the total phenolic compounds in the solution. Asdemonstrated in Table 1, hydroxytyrosol comprises over 80% of thephenolic compounds in the solution after 12 months.

TABLE 1 Conversion from Oleuropein to Hydroxytyrosol Following theAddition of About 25 Pounds of Citric Acid/1000 Gallons Composi-Composi- Composi- Composi- tion at tion at tion at tion at Component T =2 mo. T = 3 mo. T = 4.5 mo. T = 12 mo. Hydroxytyrosol 30.4%  32% 48.4%80.2%  Tyrosol 2.5%  5%  2.2% 3.6% Oleuropein  41% 36.6%  25.1% 1.2%Oleuropeinaglycone 4.2% 4.6%   2.7% 3.7%

Example 2 Conversion from Oleuropein to Hydroxytyrosol Following theAddition of About 50 Pounds of Acid/1000 Gallons

Table 2 shows the conversion of oleuropein to hydroxytyrosol over timefollowing the addition of about 50 pounds of citric acid per 1000gallons of vegetation water. The percentages in Table 2 are shown asweight percentages of the total phenolic compounds in the solution.Significantly, as demonstrated in Table 2, hydroxytyrosol comprises over45% of the phenolic compounds in the solution after 2 months.

TABLE 2 Conversion from Oleuropein to Hydroxytyrosol Following theAddition of About 50 Pounds of Acid/1000 Gallons Composi- Composi- tionat tion at Component T = 2 mo. T = 12 mo. Hydroxytyrosol 45.7% 78.5% Tyrosol  2.9% 3.3% Oleuropein 28.7% 1.5% Oleuropeinaglycone  4.1% 3.5%

Example 3 Extraction of Hydroxytyrosol from Vegetation Water

An aliquot (0.5 ml) of vegetation water containing about 40 mg of drysolid (maltodextran) was mixed with polypropylene porous beads anddried. The dry mix was used for extraction with supercritical carbondioxide (PoroCrit, LLC, Berkeley, Calif.). The collected sample (about2.0 mg) was analyzed by HPLC. The profile of the sample is shown in FIG.2 and Table 3 shows the area under the major peak to be 97%. Whensynthetic hydroxytyrosol was added to the sample and analyzed by HPLC,one major peak appeared, as shown in FIG. 3, indicating that the majorproduct of the sample is hydroxytyrosol (Table 4).

TABLE 3 Peak Analysis of FIG. 2 HPLC Results Peak No. Time Height (μV)Area (μV-sec) Area (%) 1 5.935 215542 6687705 97.476 2 11.433 5686173104 2.523

TABLE 4 Peak Analysis of FIG. 3 HPLC Results Peak No. Time Height (μV)Area (μV-sec) Area (%) 1 2.875 1345 13895 0.26 2 3.278 1076 14140 0.2653 6.641 211204 5241105 98.240 4 11.961 2587 65811 1.233

Example 4 Extraction of Hydroxytyrosol from Acidified Vegetation Water

An aliquot (1 liter) of vegetation water after acidic hydrolysis wasvigorously shaken with ethyl acetate in a shaking flask. The organicsolvent was separated from the aqueous solution and evaporated off byrotory evaporator. The resulting thick oil (about 20 g.) was collectedand analyzed by HPLC. The profile of this sample is shown in FIG. 5, andTable 5 shows the area of the major peak to be 97.457% indicating thathydroxytyrosol represents about or more than 95% of the totalpolyphenolic fraction in the water. Total phenolic determination bystandard colorimetric assay shows that the hydroxytyrosol is containedin the oil at approximately 20% in weight.

TABLE 5 Peak Analysis of FIG. 5 HPLC Results Peak No. Time Height (μV)Area (μV-sec) Area (%) 1 3.873 7620 46501 2.542 2 13.575 95112 178279397.457

This fraction was used for further purification of hydroxytyrosol by gelchromatography. Dry silica (150 g) was suspended in ethyl acetate (300ml) to obtain a thick slurry. The slurry was poured into a glass columnand the silica was allowed to stand for 15 minutes to sediment. Thethick oil containing about 20% (4 g) hydroxytyrosol was dissolved in 25ml of ethyl acetate and slowly poured over the silica gel. Thepurification of the hydroxytyrosol was obtained by gravity elution ofthe product and by the addition of ethyl acetate as the solvent. Thefractions containing the pure hydroxytyrosol were collected and pooledtogether. The solvent was evaporated until a yellow oil was produced. Asshown in FIG. 6 and in Table 6, this oil is essentially purehydroxytyrosol (97-99%) as verified by HPLC and mass spectroscopy. Theyield of this purification is about 2.8-3.0 g. Hydroxytyrosol or ca.65%.

TABLE 6 Peak Analysis of FIG. 6 HPLC Results Peak No. Time Height (μV)Area (μV-sec) Area (%) 1 2.875 1345 13895 0.26 2 3.278 1076 14140 0.2653 6.641 211204 5241105 98.240 4 11.961 2587 65811 1.233

Mass spectrometry analysis of the samples obtained as described by thetwo procedures in Examples 3 and 4, as shown in FIG. 7, confirmed thatthe major product is hydroxytyrosol. The sample was diluted to a finalconcentration of 26 micrograms per milliliter with methanol and analyzedin negative ionization mode on a Finnigan LCQ fitted with an ESI probe.The infusion was at 3 microliters per minute using an integrated syringepump. The temperature was 270 C, needle voltage +4.2 V, sheath gas 45units, and auxiliary gas 10 units. The fragmentation pathway ofhydroxytyrosol is shown in FIG. 8. As can be seen in FIG. 7,hydroxytyrosol (mass/charge 153.1) and its fragmentation products (123.1and 105.1 mass/charge) account for the majority of the product abundancein the multi-stage spectrum.

Example 5 Protection Against Skin Damage from UltraViolet RadiationOlivenol Compositions

The test article was Olivenol (Lot #1A-1B). Olivenol is the crude waterpreparation obtained by acidic hydrolysis of vegetation water (500 ml)evaporated to dryness by rotory evaporator and subsequentlyophilization. The test article vehicles were aqueous 0.5% w/vmethylcellulose (oral administration) and methyl alcohol, 99.9% A.C.S.spectrophotometric grade (topical administration). Formulations wereprepared once during the study and the test article was considered 75%active for the purpose of dosage calculations.

Mice

One hundred male Crl:SKH1-hrBR hairless mice (Source: Charles RiverLaboratories, Inc., Portage, Mich. USA) were randomly assigned to tendosage groups (Groups 1 through 10), ten mice per group as indicated inTable 1. The body weights of male mice ranged from 17 to 28 grams.

TABLE 5 Experimental Design Frequency of Formulation Formulation andDosage Concentration Route of Number of Administration Group(mg/kg/day)* (mg/mL)* Administration Male Mice (Days) 1 0 (Vehicle) 0(Vehicle) Oral 10 31 2 Olivenol (1) 0.1 Oral 10 31 3 Olivenol (10) 1.0Oral 10 31 4 Olivenol (100) 10.0 Oral 10 31 5 Olivenol (100) 10.0 Oral10 10 6 0 (Vehicle) 0 (Vehicle) Topical 10 31 7 Olivenol (1) 0.25Topical 10 31 8 Olivenol (10) 2.50 Topical 10 31 9 Olivenol (100) 25.00Topical 10 31 10 Olivenol (100) 25.00 Topical 10 10 *Groups 1-5: dosagevolume = 10 mL/kg. Groups 6-10: dosages and concentrations assume amouse body weight of 25 grams and an administration volume of 0.1mL/mouse (i.e., 100 mcL/mouse).

Administration of Olivenol Compositions and UVR Exposure

Formulations were orally administered (via gavage) to appropriate miceonce daily for either 31 (Groups 1 through 4) or 10 (Group 5)consecutive days. Formulations were topically administered (100mcL/mouse) to appropriate mice once daily for either 31 (Groups 6through 9) or 10 (Group 10) consecutive days.

On the 28^(th) day (Groups 1 through 4 and 6 through 9) or the 7^(th)day (Groups 5 and 10) of formulation administration, mice in Groups 1through 10 were exposed to UVR (i.e., wavelengths in the UVB and UVAportions of the electromagnetic spectrum). The source of irradiation wasa Berger Compact Arc high intensity solar simulator (Solar LightCompany, Philadelphia, Pa.) with a WG320 Schott glass filter (1 mm)coupled to an Oriel light pipe. The radiant intensity of the source wasmonitored continuously with a PMA 2100 meter (Solar Light Company,Philadelphia, Pa.) or comparable device. On day 28 or 7, the intervalbetween the formulation administration and the start of UVR exposure wasless than 15 minutes for most mice and slightly more than 15 minutes fora small number of mice.

Checks for viability were made twice daily. Clinical observations wererecorded at least weekly, including once before the first formulationadministration and once immediately before UVR exposure. Clinicalobservations were also recorded at approximately 24, 48 and 72 hoursafter irradiation. Body weights were recorded once weekly during theadministration period and at terminal sacrifice.

Sacrifice of Mice

All mice survived to schedule sacrifice. Scheduled sacrifice occurredafter the final examination, approximately 72 hours after completion ofthe UVR exposures (CO₂ asphyxiation). Dorsal skin, including the UVRexposure sites, were removed and retained in neutral buffered 10%formalin for possible histopathological examination.

Calculated mean UVR dose values (MED) and standard deviations weredetermined for appropriate groups as follows. The lowest instrumentalUVR dose to cause any cutaneous response at a site of exposure wasdetermined for each mouse. The mean calculated UVR dose for each groupfor each observational time point was determined. If administration ofthe test article has no influence on the UVR dose required to elicitcutaneous responses, based on this method of calculation, a meancalculated UVR dose value equivalent to 1.0 MED would be expected at 48hours after irradiation. A mean calculated UVR dose value greater than1.0 would indicate a protective effect of the test article. For anymouse that had no skin reactions in any of the six UVR exposure sites,an imputed value of 2.8 was assigned for the purpose of calculation andthe > symbol was included as a prefix to the group mean calculated UVRdose value. Additionally, ratios (clinical observations) and averageswith standard deviations (body weights) were calculated.

Group means and standard deviations were calculated and tabulated forbody weights and body weight changes.

Results

Skin reactions that occurred in the UVR exposure sites includederythema, edema and flaking and the severity of the skin reactionstended to be dependent on the UVR exposure dose.

There was an indication of a mild dosage-dependent protective effectagainst UVR-induced cutaneous inflammation in hairless mice orallyadministered Olivenol for 31 days and a moderate dosage-dependentprotective effect in mice topically administered Olivenol for 31 days.In this type of study, it was anticipated that the mean calculated UVRdose value would be approximately equal to 1.0 at 48 hours after UVRexposure in naïve mice.

In mice orally administered Olivenol for 31 days (Groups 1 through 4),the mean calculated UVR dose values at 48 hours after UVR exposure were1.2, 1.3, 1.4 and 1.5 in the 0 (Vehicle), 1, 10 and 100 mg/kg/day dosagegroups, respectively. In mice topically administered Olivenol for 31days (Groups 6 through 9), the mean calculated UVR dose values at 48hours after UVR exposure were 1.5, 1.5, >1.9 and >2.2 in the 0(Vehicle), 1, 10 and 100 mg/kg/day dosage groups, respectively. The >symbol was included as a prefix to the mean calculated UVR dose valuesin Groups 8 and 9 because no cutaneous reactions occurred in any of theUVR exposure sites for two mice in each of those groups. For those fourmice an imputed value of 2.8 was assigned for the purpose ofcalculation.

At 72 hours after UVR exposure, the protective effect of the testarticle was less definitive. However, in mice topically administered 100mg/kg/day Olivenol for 31 days (Group 9) the mean calculated UVR dosewas 1.6 at 72 hours after UVR exposure, as compared with a value of 1.2for the appropriate control group (Group 6).

The 1.5 mean calculated UVR dose value in Group 6 [0 (Vehicle), topicaladministration] at 48 hours after UVR exposure was unanticipated. Sincethe value was substantially greater than the anticipated value ofapproximately 1.0, the vehicle may have had some impact on cutaneoussusceptibility to UVR exposure. However, there was a clear increase inthe mean calculated UVR dose values in the mice topically administered10 and 100 mg/kg/day Olivenol dosages for 31 days, as compared with micetopically administered 0 (Vehicle) mg/kg/day Olivenol.

There was no indication of a protective effect against UVR-inducedcutaneous inflammation in hairless mice administered the 100 mg/kg/dayOlivenol dosage for 10 days vial the oral (Group 5) or topical (Group10) route, as compared with the appropriate control groups. In miceorally or topically administered the 100 mg/kg/day Olivenol dosage for10 days, the mean calculated UVR dose values at 48 hours after UVRexposure were 1.0 and 1.3, respectively. These values were comparable tothe values that occurred in the appropriate 0 (Vehicle) mg/kg/day dosagegroups (i.e., Groups 1 and 6, respectively).

The skin reactions that occurred at 24 hours after UVR exposure were notconsidered useful in making a determination on the protective potentialof the test article because these reactions tend to be less reproduciblethan those that occur later.

Two mice in each of Groups 1 and 3 developed urogenital ulcerations. Onemouse in each of Groups 6 and 7 developed lump(s). These are commonfindings in male hairless mice and were not considered testarticle-related.

Necropsy revealed that all tissues appeared normal.

Body weight and body weight changes observed throughout the experimentalprotocol were unremarkable.

There was an indication of a mild dosage-dependent protective effectagainst UVR-induced cutaneous inflammation in male hairless mice orallyadministered Olivenol for 31 days and a moderate dosage-dependentprotective effect in mice topically administered Olivenol for 31 days.The high Olivenol dosage, 100 mg/kg/day, afforded cutaneous protectionvia the oral and topical administration routes.

There was no indication of a protective effect against UVR-inducedcutaneous inflammation in hairless mice administered the 100 mg/kg/dayOlivenol dosage for 10 days via the oral or topical route.

In light of the detailed description of the invention and the examplespresented above, it can be appreciated that the several aspects of theinvention are achieved.

It is to be understood that the present invention has been described indetail by way of illustration and example in order to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Further, the specific embodiments of the present inventionas set forth are not intended as being exhaustive or limiting of theinvention, and that many alternatives, modifications, and variationswill be apparent to those skilled in the art in light of the foregoingexamples and detailed description. Accordingly, this invention isintended to embrace all such alternatives, modifications, and variationsthat fall within the spirit and scope of the following claims. Whilesome of the examples and descriptions above include some conclusionsabout the way the invention may function, the inventors do not intend tobe bound by those conclusions and functions, but puts them forth only aspossible explanations.

1. A method for protecting skin against adverse effects of exposure toultraviolet radiation (UVR) comprising orally administering to a subjectin need of such protection a pharmaceutically effective amount of atreatment agent having a weight ratio of hydroxytyrosol to oleuropein ofbetween about 1:1 and about 200:1.
 2. The method of claim 1, whereinsaid weight ratio is between about 4:1 and about 100:1.
 3. The method ofclaim 1, wherein said weight ratio is between about 10:1 and about 50:1.4. The method of claim 1, wherein said agent further comprises asunscreen for topical applications.
 5. The method of claim 1, whereinsaid subject is a human.
 6. The method of claim 1, wherein said agent isprepared by a process comprising the steps of: (a) producing vegetationwater from olives; (b) adding acid to the vegetation water in an amounteffective to produce a pH between about 1 and about 5; (c) incubatingthe acidified vegetation water until at least 75% of oleuropeinoriginally present in the vegetation water has been converted tohydroxytyrosol.
 7. The method of claim 6, wherein said agent is dried toprovide a powder extract.
 8. The method of claim 7, wherein said agentis in the form of a tablet, capsule, or pill.
 9. The method of claim 6,wherein said agent is in the form of a liquid.
 10. A method forprotecting skin against adverse effects of exposure to ultravioletradiation (UVR), comprising orally administering to a subject in need ofsuch protection a pharmaceutically effective amount of substantiallypurified hydroxytyrosol or a substantially purified mixture ofhydroxytyrosol and oleuropein.
 11. An oral composition comprising olivepolyphenols in an amount effective to scavenge free radicals whereinsaid olive polyphenols comprise a weight ratio of hydroxytyrosol tooleuropein of between about 1:1 and about 200:1.
 12. The oralcomposition of claim 11, wherein said weight ratio is between about 4:1and about 100:1.
 13. The oral composition of claim 12, wherein saidweight ratio is between about 10:1 and about 50:1.